The overall mission of our project is to understand the function and mechanism of complex biological assemblies by investigating their three-dimensional spatial architectures at the highest possible resolutions. We take an interdisciplinary approach to this problem, using emerging technologies in cryo-electron microscopy as our primary tool. Members of our group work on different, yet, complementary aspects of structural analysis. These include (a) electron crystallographic studies of two-dimensional membrane protein crystals, (ii) "single particle" approaches to analyze the structures of multi-protein complexes and (iii) determination of the structures of large subcellular assemblies and whole cells using electron tomography. A significant fraction of our research effort is devoted to developing and implementing novel methods for specimen preparation, high throughput data acquisition and computational analysis. The nature of our work requires expertise in areas spanning from cell and molecular biology to computational biology, physics and engineering. The research background of members in our team reflects this interdisciplinary mix, and we also benefit greatly from collaborations with a variety of scientists both inside and outside NIH. Research overview 2003-2004: Highlights from selected projects are summarized below: (i) Structural biology of membrane proteins: We are exploring the structural basis underlying the transport of ions and solutes across biological membranes using high resolution electron crystallography at resolutions in the 3 to 6 range. Recent projects have included studies of the mechanism of proton pumping by bacteriorhodopsin, mechanism of oxalate transport by the 12-helix oxalate transporter. In both cases we have deduced the mechanism of transport by determining sructures of the proteins in two different conformations with accessibility to alternate sides of the bilayer membrane. (ii) Structural biology of signal transduction in bacterial chemootaxis: A variety of projects are underway to explore fundamental mechanisms underlying signaling mechanisms in prokaryotic and eukaryotic cells. For example, we are using electron tomography and single particle electron microscopy to analyze the spatial architecture of the signaling apparatus used by bacteria to sense and respond to extracellular ligands. We are studying the molecular structure of multiprotein complexes involved in cellular metabolism and the large complexes that serve as rotary machines to support motion of bacterial flagella. (iii) Structure of HIV and the mechanism of cellular entry: Several projects are underway that relate to structural exploration of the 3D architecture of HIV and the fundamental mechanisms involved in virus-cell contact and ensuing entry using a combination of electron tomography and single particle approaches. (iv) Development of technology for automated electron microscopy: We have continued our development of tools for automation in data collection and image processing with implementation of a new 100-specimen stage holder for high throughput electron microscopy (in collaboration with GATAN Inc.) and the development of a new methodology for automated 3D imaging of tissue using a new generation of dual-beam electron microscopes (in collaboration with FEI Inc.).